Method and apparatus to mitigate evaporation in high throughput measurements

ABSTRACT

A method and apparatus are disclosed for the collection of light scattered from a liquid sample contained within a multiwell plate for which evaporation from the wells is mitigated by the application of a barrier between the liquid sample and the environment. A vertical thermal gradient is applied across the vessel so that condensation is inhibited from forming on the interior surface of the barrier, thus permitting clear illumination of the sample for visual imaging, fluorescence studies and light scattering detection.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/727,521, filed Oct. 6, 2017.

RELATED APPLICATIONS AND PATENTS

The following patents relate to the measurement of the physicalproperties of liquid samples in a multiwell plate and are herebyincorporated by reference:

U.S. Pat. No. 6,519,032 B1, S. C. Kuebler and J. Bennett, “Fiber opticapparatus and use thereof in combinatorial material science,” issuedFeb. 11, 2003.

U.S. Pat. No. 6,819,420 B2, S. C. Kuebler and J. Bennett, “Fiber opticapparatus and use thereof in combinatorial material science,” issuedNov. 16, 2004.

U.S. Pat. No. 8,964,177 B2, D. I. Some, M. I. Larkin, P. G. Neilson, andD. N. Villalpando, “Method and apparatus to illuminate sample andcontaining vessel in a light scattering detector,” issued Feb. 24, 2015.

U.S. Pat. No. 8,976,353 B2, M. I. Larkin, A. D. Hanlon, D. I. Some, R.J. Sleiman, D. N. Villalpando, “Multiwell plate lid for improved opticalmeasurements,” issued Mar. 10, 2015.

U.S. Pat. No. 9,347,869 B2, M. I. Larkin, A. D. Hanlon, D. I. Some, R.J. Sleiman, D. N. Villalpando, “Multiwell plate lid for improved opticalmeasurements,” issued May 24, 2016.

U.S. Pat. No. 9,658,156 B2, A. D. Hanlon, M. I. Larkin, “Method ofcharacterizing interactions and screening for effectors,” issued May 23,2016.

U.S. Pat. No. 9,459,207 B2, A. D. Hanlon, M. I. Larkin, “Method ofcharacterizing interactions and screening for effectors,” issued Oct. 4,2016.

BACKGROUND

Throughout this specification, the term “particle” refers to theconstituents of liquid sample aliquots that may be molecules of varyingtypes and sizes, nanoparticles, virus like particles, liposomes,emulsions, bacteria, colloids, etc. Their size range may lie between 1nm and several thousand micrometers.

Light scattering is a non-invasive technique for characterizingmacromolecules and a wide range of particles in solution. The two typesof light scattering detection frequently used for the characterizationof macromolecules are static light scattering (SLS) and dynamic lightscattering (DLS).

Static light scattering experiments involve the measurement of theabsolute intensity of the light scattered from a sample. Thismeasurement allows the determination of the size of the samplemolecules, and, when coupled with knowledge of the sample concentration,allows for the determination their weight average molar mass. Inaddition, nonlinearity of the intensity of scattered light as a functionof sample concentration may be used to measure interparticleinteractions and associations.

Dynamic light scattering is also known as quasi-elastic light scattering(QELS) and photon correlation spectroscopy (PCS). In a DLS experiment,time-dependent fluctuations in the scattered light signal are measuredusing a fast photodetector. DLS measurements determine the diffusioncoefficient of the molecules or particles, which can in turn be used tocalculate their hydrodynamic radius.

Extensive literature has been published describing methods for makingboth static and dynamic light scattering measurements in flowing andbatch (non-flowing) systems. See, for example, P. J. Wyatt, “Lightscattering and the absolute characterization of macromolecules,”Analytica chimica Acta, 272, 1-40, (1993). Many commercially availableinstruments allow for the measurement of SLS and/or DLS, and there aremany methods to perform these measurements. For example, U.S. Pat. No.6,819,420, by Kuebler and Bennet, discloses a method and apparatus formeasuring the light scattering properties of a solution in a vesselwherein light may be transmitted into the solution through the bottom ofthe optically transparent vessel, and the scattered light may bedetected through the same surface by means of an optical fiber coupledwith a photodiode.

With the development and improvement in the optical quality of multiwellplates, it has become possible to make both SLS and DLS, as well asother measurements of the physical properties, such as fluorescence,concentration, and absorption, directly from samples contained therein.Methods capable of measuring samples directly in these multiwell platesare generally desirable given both the high-throughput nature of themeasurements and the reduced sample volume requirements. Multiwellplates may contain any number of independent wells. Most commerciallyavailable plates for analyses such as these contain either 96, 384, or1536, each well is able to contain a different sample, and all wells maybe tested in a single data collection run. In addition, use of theseplates obviates the laborious need to clean and dry individualscintillation vials after each measurement. These plates generally havevery low volume wells, and commercially available multiwell plate basedmeasurement instruments are capable of light scattering measurementsfrom sample volumes of 4 μL or less. These tiny sample volumes are ofgreat benefit when one has a limited amount of sample from which to makemeasurements, particularly when compared to the 300 μL or larger sizedmeasurement volumes often required by other light scattering techniques.

All light scattering measurements are subject to various sources ofunwanted noise, which can lead to inaccurate measurements of the lightscattering properties of the sample itself. This noise may be due tounknown contaminants present in the sample, soiled or improperlymanufactured or maintained or dirty surfaces of the vessel through whichthe light transmitted and/or measured passes. Imperfections in thesurfaces of the vessel or other contaminants contained therein oradhered thereto, such as bubbles, precipitated particles, residue, etc.,may also cause background scattering which can also interfere withproper measurements of scattered light from the sample or may interferewith the beam or scattered light expected to exit the vessel and bemeasured by a detector. In other words, deleterious high backgroundsignal, or noise, is caused by light scattered from anything other thanthe sample. This background noise decreases the light scatteringinstrument's sensitivity due to the increase in the noise present inrelation to the useful signal scattered from the sample itself, andtherefore an overall reduction in the signal-to-noise ratio upon whichthe sensitivity of the measurement is dependent. For DLS measurements,higher sample concentrations of precious sample materials are requiredto overcome this background signal.

While light scattering detection in multiwell plates has manyadvantages, including high throughput measurements, the ability tocontrol the temperature of multiple samples simultaneously, and theability to monitor aggregation and other self and hetero associations,to name only a few, there are special pitfalls associated with thesemeasurements. For example, gas bubbles may adhere to the bottom or sideof the well, or may float within the sample itself or at or near thefluid meniscus. In addition, multiwell plates may be reused, and thuscareful cleaning is required between sample collections; imperfectwashing may leave behind artifacts or residues that can deleteriouslyaffect light scattering measurements. The amount of time required of anoperator or a robotic injector to fill an entire plate opens up thepossibility for dust particles to fall into the wells or othercontaminants to be introduced thereto by the handling of the plateswhile loading wells, such as oil from skin, powder from handling gloves,cosmetics, flaking skin cells, debris from loading pipettes. In order tomitigate problems associated with evaporation, an oil overlay is oftenused to “cap” a well, and residues and/or droplets from this oil mayremain in a well. In order to identify potentially contaminated samplecell wells, Some, et. al. disclosed, in U.S. Pat. No. 8,964,177 B2, asystem whereby the multiwell plate is illuminated enabling highresolution photos of wells to be taken and stored in software withoutinterfering with light scattering measurements, and thus, when analyzingthe data, correlations can be made between data and cleanliness of thewell from which it was taken. While not eliminating the contaminants,this system helps to alleviate some of the errors associated with lightscattering measurements in multiwell plates.

Another problem associated with all so-called “batch” light scatteringmeasurements, that is, measurements taken from a static sample within aflow cell, wherein, generally, the sample is exposed at least partiallyto the environment via a sample/air interface, is the issue ofevaporation. Evaporation can alter the sample state, skew resultsthrough altered background intensity, or prohibit light scatteringmeasurement entirely. Partial evaporation of the solvent from a wellincreases the concentration of the dissolved solute, which may havedeleterious effects on the sample itself. Evaporation can also impactthe meniscus as well as meniscus height in the well, leading toinconsistent results. More substantial evaporation of the sample solventcan often completely prevent accurate measurement, which is a problemparticularly prevalent in very small volume multiwell plates where evena small amount of evaporation results in a large change in the height ofthe fluid level. Even for the larger sample volumes contained in 96 wellplates, evaporation concerns often prevent useful extended measurementtimes as well as measurements at elevated temperature, making studies oftemperature dependence exceedingly difficult.

Traditionally evaporation from well plates has been addressed by eithera film or cover placed on the surface of the plate above the samplewells or, as mentioned above, a layer of oil overlaying the samplecontained in each well. However, for light scattering measurements, bothof these commonly used evaporation mitigation techniques can beproblematic. Films and solid transparent covers can promote significantbackscatter from the interface of the exiting light beam with the lid orfilm. While this problem may be partially overcome by employing a lidthat absorbs light at the wavelength of the illumination source, otherproblems still exist. One of the largest problems associated withevaporation in covered multiwell plates concerns fluid contained in thewells evaporating and then condensing on the inner surface of the filmor cover. This layer of condensation is highly scattering and isgenerally non-uniform from well to well, and thus again, the backscatterintensity may overwhelm sample signal, greatly decreasing thesensitivity of the measurement, and often leading to erroneous results.While the use of an oil overlay eliminates the issue of condensation,the potentially negative interactions of oil and sample molecules arewell known, as documented in the 2004 article by L. S. Jones et al,“Silicone oil induced aggregation of proteins,” published in the Journalof Pharmaceutical Sciences, v. 94, pages 918-927. Such unintendedinteractions may result in an inaccurate representation of the truesample characteristics, and may occur without the knowledge of theexperimenter. In addition, oil overlays can be difficult and timeconsuming to apply to each sample containing well. The practicalrequirement of such an oil overlay to control condensation prevents manyusers from attempting multiwell plate-based light scatteringmeasurements.

Many of these issues were discussed and addressed in U.S. Pat. No.8,976,353 B2 by Hanlon, et. al. with the use of a novel lid structurewhich contained posts or tubes which protruded from the bottom lidsurface into each well of the sample. These lids sealed each wellindividually as well as provided means to direct or collect theilluminating beam after passage through the sample. However, the expenseof these specialized lid elements in addition to difficulties withcleaning between uses may prevent some researchers from employing them.Further, these specialized lid elements tend to interfere with onboardoptical cameras such as those discussed above and in U.S. Pat. No.8,964,177 B2, which image the contents of the wells.

It is an objective of the present invention to offer a simple, userfriendly means to mitigate many of the problems associated withevaporation from samples in multiwell plates without the need for oiloverlays, which can be burdensome and may interact with samples underinvestigation, as well as offering a cost effective alternative toutilizing specialized multiwell plate lids. Another objective of thepresent invention is to provide evaporation mitigation means whileretaining the ability to take high quality photographs of the wells of amultiwell plate contained within a light scattering instrument.

A BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a light scattering/photographic multiwell plate reader asdescribed by in U.S. Pat. No. 8,964,177 B2 incorporating a means toprovide illumination for photographs.

FIG. 2 illustrates critical elements of an embodiment of the inventionincluding a heating source to generate a temperature gradient across thecollection enclosure as well as across the sample plate itself.

FIG. 3 shows a preferred embodiment of the invention with elements tomitigate evaporation and condensation while enabling collection ofphotographic and light scattering data from the sample wells.

A DETAILED DESCRIPTION OF THE INVENTION

Another common use for multiwell plates where evaporation can be aconcern is in the biochemical field of polymerase chain reaction (PCR)experiments. In a typical PCR experiment, the reaction of a liquidsample of proteins, for example, are exposed to varying amounts of aparticular enzyme placed within individual conical vials, each with itsown incorporated lid, and these vials are placed within a single plateplaced within the walls of a multiwell plate, generally adapted toreceive the conical vials. The properly loaded multiwell plate thenundergoes thermal cycling, generally by being placed within a chamberthat cycles between approximately 96° C. for the denaturation step anddown to approximately 60° C. for the annealing step. This cycling isgenerally repeated between 20 and 40 times. As with light scatteringapplications, evaporation can be a problem in PCR analysis. Means bywhich the PCR samples are heated and cooled can vary depending on theinstrumentation. In some cases, the vials or strips of vials arecontained within a heat conducting plate, which is used to raise andlower the temperature. In these cases a top plate is often placed intocontact with the lids of the vials. Throughout the cycling procedurethere is a risk of evaporation from the sample, resulting incondensation of the solvent upon the inner lid of each vial. Thisevaporation necessarily changes the concentration remaining sampleconcentration contained within the main body of the vial, and over theentire cycling process, these variations in sample can affect results.Further, some of the techniques used to heat the chamber, for example,external heaters, can result in uneven heating throughout the chamber,expansion of the retaining plate, and uneven evaporation across thesamples contained therein, thus some vials may experience evaporationinduced concentration change, while others may have more significantchanges, and thus uniformity of results are not guaranteed. In addition,as the plates are heated by a thermal block within the retaining bottomplate and a heated lid located just above and/or in contact with thesample lids, the wells can expand and introduce small spaces between thewell walls and the cap/sealing means where gasses can escape. Theseexpansion issues are partially mitigated by two-piece constructionplates such as the Framestar™ plates by 4titude® (Wotton, Surrey, UK)that utilize polypropylene tubes and polycarbonate holding frames,though even these advanced systems still result in some lost samplevolume by evaporation.

While evaporation concerns pose challenges to both PCR and lightscattering detection, it should be noted that PCR is primarily apreparative technique, and thus the methods for dealing with evaporationin PCR can differ from methods utilized with light scatteringmeasurements. For example Yamamoto, et. al., in European PatentPublication No. 0 311 440 B1, disclose a multi-step, small footprint,preparatory instrument for dispensing, preparing and cycling samples forsubsequent analysis by another instrument. In one region of theapparatus an automated pipette takes sample from reagent bottles placedon one stage and injects them into prepared wells of a multiwell plateon a second stage. The stage containing the multiwell plate is thenmoved, by an automated system, into the heating chamber, isolated fromthe preparative chamber, where the samples are brought to the desiredtemperature by placing a heating block in contact with the multiwellplate both above and below. In one embodiment of the Yamamato invention,when the desired temperature has been reached (and cycles throughtemperatures achieved as necessary), the bottom heating plate may beremoved from the sample, while retaining the top heating in heattransmitting contact with the multiwell plate. This method encouragesthe cooling rate of the liquid samples in the wells of the multiwellplate to be more rapid than the cooling rate of the upper heater, andtherefore liquid vaporized in the space in the well can be cooleddiscouraging the condensation of sample on the lower surface of theupper heater plate. Once the multiwell plate is at an appropriatetemperature, the upper heating plate is removed from contact with themultiwell plate, which is then moved back into the loading chamber, andeither removed manually from the instrument or further processed by theaddition of other compounds, enzymes, etc., prior to being returned tothe heating chamber for further temperature cycling. Once the samplepreparation is complete, the multiwell plate can be moved from the PCRstation to an analysis instrument which can be either one which removesthe prepared samples from the wells for analysis, for example, byinjection into an chromatography system, or they may be analyzed withinthe plate itself by any number of means, including light scattering.

However, even with these advanced, automated preparatory systems,evaporation still remains a problem when it comes to the direct in-situanalysis of the samples within the wells of the multiwell plate. Whilethe device of Yamamoto may succeed in retaining as much of the samplewithin the well as possible during preparation, there is still ampletime within the actual analysis instrument for evaporation to be aconcern. As discussed above, instruments such as the DynaPro PlateReader II (Wyatt Technology, Santa Barbara, Calif.), enable themeasurement of light scattered from samples contained within multiwellplates, however, light scattering measurement, particularly DLSmeasurements, generally take 5-10 seconds per measurement. Therefore inorder to scan a complete 1536 well plate could take several hours.During this time, it is generally important that all the wells maintainas close to their original state as possible, and any evaporationoccurring between the time the measurements of the first and last samplewill result in inaccuracies in the derived results due to the physicaland chemical changes of the sample containing well. Therefore, it is ofcritical importance that the effects of evaporation be mitigated to thelargest extent possible when performing light scattering measurements,whether or not the samples have previously undergone PCR processing.

As discussed above, a common method for discouraging evaporation fromindividual wells is to provide a physical barrier covering the top ofeach well. These methods can include specialized lids, an oil overlay oran adhesive barrier such as tape adhered to the top of the well plate.It is an objective of the present invention to enable both thephotographic imaging of the well itself and light scattering detectionof the sample while also mitigating evaporation within the multiwellplate and, in particular, condensation on any barrier placed on theplate. The prior art, including that discussed above, is incompatiblewith these goals. For example the lids proposed by Hanlon, beyond beingexpensive, will interfere with photography of the wells. The heatingplates in contact with the multiwell plate discussed by Yamamoto, aswell as those utilized in the Framestar system, will block bothillumination of the plate for photographic purposes as well lightscattering detection via a moveable probe (or moving plate relative to afixed probe or a combination thereof).

Several embodiments of the present invention utilize both an active,though optically compatible, barrier system coupled with a more passiveheating system, permitting optical access to the plate for both lightscattering and photographic probes while preventing both evaporation andinterfering condensation. FIG. 1 shows a conventional lightscattering/photographic multiwell plate, such as described by Some inU.S. Pat. No. 8,964,177 B2 which utilizes an absorbing/transmittingoptical structure 101 located above the wells of a multiwell plate 102.This absorbing/transmitting optical structure 101, absorbs light at thewavelength of the light scattering illumination source 103 generally alaser, but transmits light at other wavelengths. This light scatteringillumination source 103 generates a beam 104 at a wavelength of λ₁. Thelight scattering source beam 104 passes through the bottom of amultiwell plate 102. The light scattering beam emerges from the samplecontained within an individual well 105, and is intercepted byabsorbing/transmitting optical structure 101 located above the multiwellvessel. Light scattered from the illuminating beam 104 by the samplecontained in the well 105 is detected by one or more light scatteringdetectors 108, wherein this optical structure 101 has been selected toabsorb radiation at the wavelength of the light scattering source. Asecond image illumination source 109 emits a generally more diffuse beamat a different wavelength, λ₂, or range of wavelengths, and its beam isincident at an angle on the absorbing/transmitting optical structure101, generally made of a special glass, which is located above themultiwell plate 102. This absorbing/transmitting optical structureabsorbs light at the wavelength of the laser, λ₁, but transmits at leastsome of the light at the wavelength, λ₂, of the image illuminationsource 109. Optical filter glass that absorbs at particular wavelengthsthat may be employed as the absorbing/transmitting optical structure 101is available from optical suppliers such as Schott North America, Inc.(Elmsford, N.Y.). The transmitted light from the imaging illuminationsource is then scattered and reflected from a diffusing surface 106,which can take many forms including that of a plain piece of whitepaper, and after transmission back through the absorbing/transmittingoptical structure 101, illuminates the wells from above. Theabsorbing/transmitting optical structure 101 therefore acts as a “beamdump” for the light scattering source 103 while transmitting lightrequired for illumination at a wavelength, which the camera 107 is ableto register. It should also be noted that the image illumination source104 or an additional diffuse illumination source may act as anexcitation radiation source to induce fluorescence in one or moresamples contained within the wells of the plate 102. In this case afluorescence detector, such as a independent fluorescence probecomprising, for example, a photodiode and a means to filter light at thewavelength λ₂. In another embodiment the camera, coupled with anappropriate filter, or a second camera and filter may act as thefluorescence detector. In other embodiments, multiple probes may beutilized to measure both static and dynamic light scattering as well asfluorescence as presented in pending U.S. patent application Ser. No.15/583,899, “High throughput method and apparatus for measuring multipleoptical properties of a liquid sample,” filed May 1, 2017 by Hsieh, et.al, incorporated herein by reference. Some embodiments of the presentinvention include independent probes for the measurement of fluorescenceand light scattering, but in some embodiments, the fluorescenceexcitation source may be a diffuse source such as the image illuminationsource 104 as discussed above.

The absorbance and transmittance of the plate 101 may be chosen tocorrespond to the wavelengths of the two light sources, or vice versa,and some variation of wavelengths λ₁ and λ₂ may be possible. Forexample, a plate may be chosen which absorbs at 830 nm±30 nm, buttransmits at all other wavelengths. Therefore the wavelength of thelaser may operate anywhere within that range or on the peripheriesthereof so long as the plate adequately absorbs the beam such that lightscattering signals are not deleteriously affected and the instrument isnot damaged.

While a plain sheet of white paper may act as a diffusing surface 106,it is also possible to use any number of other surfaces, such as adiffusing plastic produced by 3M (St. Paul, Minn.), so long as they aidin the diffusion of light to be transmitted back through the absorbingplate 101. A thin weatherproof vinyl sheet has also proven very usefulas a diffusing element 106. One particular advantage of this specificelement is that the weatherproofing aids in maintaining the integrity ofthe diffusing sheet over many temperature ramping cycles, which may becommon in high throughput light scattering experiments, for example.Further the diffusing surface need not even be an additional element,but rather could be a special diffusing surface incorporated into theglass or a diffusing layer etched, adhered, or placed thereon.

While the systems discussed thus far represent an improvement on theprior art, they do not yet solve the fundamental challenge at issue inthis disclosure, control of evaporation without interfering with opticalaccess to the each well and without the deleterious effects of oilcapping each well. For most applications the use of a barrier such astransparent adhesive tape covering the mouths of the wells or a lid isacceptable, but this raises the problem of condensation on the tapeunacceptably increases the background scattering from the probe laser.The essential inventive element is to insure that each well sees avertical temperature gradient, with the top of each well, held at ahigher temperature than the bottom of the well. This prevents dropletsfrom forming on the sealing tape. It also has the side benefit ofpreventing convection within the fluid of each well.

There are several ways that the vertical temperature gradient can beapplied. The simplest is to add a heating element to the top of theinstrument that heats the air above the plate. Indeed, when theinstrument is run above ambient temperature, the measurement chamber isinsulated from the environment but regardless of how efficient is theinsulation heat will be lost through these surfaces. If the instrumentis heated from below, which is often the case, the upper surface of themeasurement chamber is naturally cooler than the interior, whichgenerates a small negative temperature gradient, which exacerbates thecondensation problem. Adding the top heater allows the instrument tooverwhelm the heat lost to the environment to generate the desirablepositive temperature gradient.

An alternative method of applying a gradient, rather than heating theair at the top of the measurement chamber is to use one or more IRilluminators to apply infrared radiation to the sealing tape. Since thetape has a small thermal mass, it can be heated rapidly, withoutsubstantially affecting the temperature of the samples within each well.Moreover, if the camera, DLS, SLS, or florescence sensors are sensitiveto infrared illumination used to create the thermal gradient, theilluminator operation can be multiplexed with the measurement sensors sothat they only apply light when the wells are not actively beingmeasured.

FIG. 2 illustrates the fundamental elements of the invention in one ofits simplest forms. The multiwell plate 201 containing liquid samples202 to be investigated. A barrier 203 such as transparent adhesive tapeis placed on the top surface of the multiwell plate 202. A vapor region204 of each well is defined as the space above the meniscus of theliquid sample 202 and the bottom of sealing barrier 203, in other words,the vapor region is the volume of each well not occupied by liquidsample. Beneath the multiwell plate 201 are placed the various opticalinvestigation probes 205, which may include an optical camera, DLS andSLS detectors and related optics, as well as fluorescence detectors. Inother embodiments, the probes located beneath the plate may be ends ofoptical fibers and associated optics as required that directillumination to the sample or plate and/or collect and direct light fromthe sample and/or plate to the detection means. Above the sealedmultiwell plate, but not in contact therewith, is a heating element 206.The purpose of the heating element is to generate a temperature gradientacross the vertical height of the multiwell plate. However, it should benoted the measurement chamber is thermostated with either a heater or aPeltier device to a constant temperature and an additional statictemperature gradient is introduced by the top heating element. In thepreferred embodiment, this is achieved with a heater on the top surface,although it is clear that a vertical thermal gradient can also beachieved by placing an additional cooling element beneath the plate.However this variation is more difficult to achieve in practice as thelight measurement activity beneath the plate interferes with generatinga uniform cooling distribution.

The desired temperature gradient across the plate ΔT_(p) is thatdifference in temperature required to keep solvent from the sample inthe gaseous state contained within the vapor region 204 of each cell inthe gaseous state, that is to say that the temperature at the barrierT_(b) is greater than the temperature of the liquid sample T_(s), thusthe gradient ΔT_(p)=T_(b)−T_(s)>0. For most aqueous samples in standardmultiwell plates, ΔT_(p) will be approximately 0.5° C. This requiredtemperature gradient may vary depending on the amount of sample in thewells, the types of sample, the solvent, the solute, the size of thewells, and other variables, however for each different sampleconfiguration, the required ΔT_(p) can be determined and applied withthe present invention. In order to achieve the required ΔT_(p), asignificantly larger temperature gradient must be applied over thevertical height of the cavity, ΔT_(v) with the heating element locatedcentimeters above the sample containing multiwell plate. So, forexample, in one preferred embodiment of the invention shown in FIG. 3,the temperature gradient across the vertical height of the well platecontaining chamber ΔT_(v) is roughly 30° C. in order to achieve thecritical ΔT_(p) value of 0.5° C.

FIG. 3 shows one preferred embodiment of the invention combining manyelements so as to maximize the amount of information about samplesanalyzed by the plate reader. This particular novel embodiment permitsmeasurement of several properties of the liquid samples while minimizingany negative issues associated with evaporation, which has heretoforenot been possible. The plate reader instrument 301 houses a measurementchamber 302 into which is placed the filled multiwell plate 303. One ormore of the wells of the multiwell plate contains a liquid sample 304.Samples to be protected against evaporation are covered with atransparent optical barrier 305. This barrier can take many forms suchas an adhesive film or tape or a lid capable of sealing each wellindividually. Under certain, specialized circumstances, such as when allsamples are of identical composition, a more general lid will suffice asa barrier, that is to say one wherein the wells share a vapor region,but the lid retains vapor from exiting the shared vapor region into themeasurement chamber 302. The barrier, however, as previously stated,must separate vapor regions associated with the multiwell plate fromthat associated with the sample chamber, preventing, thereby, theevaporation of sample solvent into the measurement chamber. In additionthe multiwell plate must be transparent to at least certain wavelengthsof light discussed below, and in most cases will be transparent to allwavelengths of visible and UV light. The measurement chamber is sealed,generally from above, by a door element 315. Housed within this doorelement, or moved into place between the door and the plate when thedoor is closed, is one or more heating elements 306. These heatingelements may independently provide uniform heat distribution across agiven region or a heat distributor, such as a metal block 307 may beconnected thereto to effective and efficiently distribute the heatapplied by the heating elements 306. In order to permit the collectionof photographic data, as discussed above, the plate 303 is preferablyilluminated relatively uniformly. An absorbing/transmitting opticalelement 308 that absorbs light at certain wavelengths and transmits atothers is positioned beneath the heat distributor 307. Optical filterglass which absorbs at particular wavelengths and which may be employedas the absorbing/transmitting optical structure are available fromoptical suppliers such as Schott North America, Inc. (Elmsford, N.Y.).One or more diffuse illumination sources 309 are positioned within themeasurement chamber and directed to strike the absorbing/transmittingoptical element. The optical properties of the absorbing/transmittingelement are selected such that it transmits light at one or more of thewavelengths emitted by the diffuse illumination sources 309, but absorbslight at the wavelength of the light scattering source 310 discussedbelow. In a preferred embodiment light from the diffuse illuminationsource passes through the absorbing/transmitting element and isscattered from a diffusing surface 311, such as a plain piece of whitepaper. The light scattered by this diffusing surface is transmitted backthrough the absorbing/transmitting element 308 and illuminates the wellsfrom above.

Positioned beneath the multiwell plate are one or more detector elementsand accompanied illumination elements. In particular embodiments, alight scattering source 310 with a wavelength selected in the absorbingrange of the absorbing/transmitting element 308 illuminates the sample304 contained within a given well of the multiwell plate. The beam,emerging from the sample ultimately intersects theabsorbing/transmitting element and is absorbed thereby. One or morelight scattering detectors 312 collect light scattered by theilluminated sample. A camera element 313, also generally located beneaththe well plate and that may be on the same movable stage as the lightscattering illumination and detection means, takes photographic data ofthe plate and sample when the plate is illuminated by the diffuseillumination sources 309. Other detectors and illumination sources, forexample ultraviolet (UV) light sources and fluorimeters may accompanythe light scattering detector and illumination means beneath themultiwell plate. Further, it may be advantageous under certaincircumstances to have the diffuse illumination sources themselvesoperate at multiple wavelengths or be accompanied by other diffuseillumination sources whereby diffuse illumination generated by thesesources both enable photographic data to be collected by the cameraelement 313 as well as provide ultraviolet, excitation illumination tofluorescing samples. Variable wavelength photodiodes may be utilized ora plurality of diffuse light sources including those in the visible andin the UV spectrum may be utilized.

As discussed previously, in order for a temperature gradient of adequatemagnitude to be applied across the height of the multiwell plate,generally of approximately 0.5° C., a much larger temperature gradientis required over the height of the measurement chamber 302. In somepreferred embodiments this gradient must be approximately 30° C. Inorder to efficiently provide this temperature gradient without excessiveloss of heat to the environment outside of the plate reader instrumentitself, it is generally important to provide adequate thermalinsulation.

Accordingly in insulation element 314 may be placed above the heatingelements 306 in the lid element 315, thereby conserving the heatgenerated by the elements and reducing the amount of heat loss into theenvironment. Aerogels are an ideal insulator for this purpose as it ishas a low density and low thermal conductivity, and they may be customformed. In addition to heat escaping from above the heating elements,the entire measurement chamber should be appropriately insulated andvented such that the vertical gradient across the measurement chambermay be maintained. With this gradient maintained once the vapor pressurein the vapor region of the sealed well equilibrates as long as thenecessary thermal gradient is maintained, evaporation from the sample iseliminated and condensation on the sealing film is prevented, permittingphotographic as well as light scattering and other optical measurementsheretofore not possible.

There are many embodiments of our invention that will be obvious tothose skilled in the arts of measurement optics and fluid dynamics thatare but simple variations of our basic invention herein disclosed thatdo not depart from the fundamental elements that we have listed fortheir practice; all such variations are but obvious implementations ofthe invention described hereinbefore and are included by reference toour claims, which follow.

What is claimed is:
 1. An apparatus comprising: a barrier configured tobe positioned above at least one well, wherein the at least one well isconfigured to contain a liquid sample, wherein a vessel comprises the atleast on well, wherein the vessel is transparent and is configured to beplaced within a measurement chamber, wherein a light measurementapparatus comprises the measurement chamber, wherein the lightmeasurement apparatus is configured to measure light scattered from theliquid sample and to collect the light scattered from the liquid samplefrom below the vessel, wherein the barrier is configured to seal the atleast one well from the measurement chamber; and at least one heaterpositioned on an upper surface of the measurement chamber, wherein theat least one heater is configured to create a temperature gradientbetween the sample and an internal surface of the barrier to preventformation of condensation on the internal surface.
 2. The apparatus ofclaim 1 wherein the barrier is adhesive tape.
 3. The apparatus of claim1 wherein the barrier is a transparent lid.
 4. The apparatus of claim 1wherein the at least one heater is an infrared illuminator.
 5. Theapparatus of claim 4 wherein the infrared illuminator is a flash lamp.6. The apparatus of claim 1 wherein the vessel is a multiwell plate. 7.The apparatus of claim 1 wherein the temperature gradient is greaterthan 0.5° C.
 8. A method comprising: positioning a barrier above atleast one well, wherein the at least one well is configured to contain aliquid sample, wherein a vessel comprises the at least on well, whereinthe vessel is transparent and is configured to be placed within ameasurement chamber, wherein a light measurement apparatus comprises themeasurement chamber, wherein the light measurement apparatus isconfigured to measure light scattered from the liquid sample and tocollect the light scattered from the liquid sample from below thevessel, wherein the barrier is configured to seal the at least one wellfrom the measurement chamber; and positioning at least one heater on anupper surface of the measurement chamber, wherein the at least oneheater is configured to create a temperature gradient between the sampleand an internal surface of the barrier to prevent formation ofcondensation on the internal surface.
 9. The method of claim 8 whereinthe vessel is a multiwell plate.